Malignant gliomas are the most common primary central nervous system (CNS) malignancy in adults, responsible for >14,000 deaths in the U.S. in 20122. The World Health Organization (WHO) has established a number of histologic and clinical criteria used for classifying gliomas into various subtypes and grading them I to IV, indicating their degree of malignancy. Diffuse gliomas (WHO grade II-IV), which include astrocytomas, oligodendrogliomas, oligoastrocytomas, and glioblastomas (GBM)3 are of particular clinical importance as they account for 80% of all primary malignant brain tumors. These tumors are diffusely infiltrative, which makes curative surgical resection impossible. Additionally, grade II-III diffuse gliomas also have the ability to progress to higher WHO grade IV GBM. GBM is the most common malignant brain tumor in adults and has the worst survival (median overall survival 12-15 months)4. Additionally, even among entities with identical histology, patient outcome can vary substantially. This is best exhibited by primary GBM, which occurs de novo as compared to secondary GBM, which progresses from lower grades. Both tumors histologically are indistinguishable, but genetically and clinically these diseases are distinct, as the survival of patients with secondary GBM is almost double that of primary GBM5.
Accurate diagnosis of diffuse glioma is particularly challenging due to heterogeneity, invasiveness, reactive parenchyma, and ambiguity among morphologic features. These diagnostic challenges are reflected by the high degree of inter-observer variability seen in clinical use of these criteria. In a study of 244 gliomas reviewed independently by four neuropathologists, concordance rates were as low as 52%6. Accurate diagnosis of diffuse glioma is critically important for clinical decision-making for patients. This diagnosis determines the treatment regimen, and particular subtypes are known to show increased treatment response to particular chemotherapies (e.g., procarbazine, CCNU, and vincristine for oligodendroglioma treatment). Additionally, histologic subtype dictates patient prognosis. Objective, tumor specific markers are clearly needed for more accurate diagnosis, prognosis and delivery of personalized care to glioma patients.
To address these needs, large-scale sequencing studies have profiled the genetic alterations found in diffuse glioma. Many alterations were noted, such as frequent mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2)1, the promoter of telomerase reverse transcriptase (TERT)7, alpha thalassemia mental retardation syndrome X-linked (ATRX)8,9, homolog of Drosophila capicua (GIG), far upstream element binding protein 1 (FUBP1)10, among others. These findings have helped to establish clear objective molecular subtypes of glioma. In terms of relevance to diagnosis, alterations in the TERT promoter and IDH1/2 are the most promising due in large part to their frequency and their occurrence as single nucleotide substitutions at specific genomic loci (“hotspots”). In diffuse glioma, we found that the degree to which these mutations co-occur or occur exclusively defines glioma subtypes: e.g., IDH1/2 mutations occur in >50% of secondary GBMs but are infrequent in primary GBMs (<5%) (FIG. 1), while TERT promoter mutations are found in >80% of primary GBMs as well as in >70% of oligodendrogliomas (FIG. 2). Furthermore, we found that the genetic subtypes of glioma established by TERT/IDH status effectively stratifies glioma patients into subtypes with distinct prognoses, more effectively than by histology alone, providing physicians with an objective test to guide more appropriate treatment (FIG. 3). For example, patients with gliomas harboring TERT promoter mutations have median overall survival (OS) of 11.5 months, while those with both TERT promoter and IDH1/2 mutations exhibit a median OS of 125 months. Additionally, our studies have revealed that the “mixed histology” oligoastrocytomas, which likely reflect the ambiguity of histologic classification most, genetically stratify into either a astrocytic (TERTWTIDHMUT) or oligodendroglial (TERTMUTIDHMUT) signature (FIG. 3). We have expanded our study of these mutations to a number of other tumor types and found that TERT promoter mutations are also frequent in other cancers, most notably liver cancer (44.2%), bladder cancer (66%), myxoid liposarcoma (79.1%), and medulloblastoma (21%), in which early diagnosis, recurrence and therapeutic response monitoring is critical7,11. (FIG. 2) Similarly, IDH1 and IDH2 mutations have been found in high frequencies in other types of cancers as well, including chondrosarcoma (56%)12, enchondroma (87%), spindle cell hemangioma (70%)13,14, acute myeloid leukemia (15%)15, and cholangiocarcinoma of intrahepatic origin (22-28%)16.
These glioma subtype-specific and highly recurrent mutations call for diagnostic assays that are able to rapidly, sensitively, and specifically detect these mutations in IDH1/2 and the TERT promoter. Such a tool would aid neuropathologists in these challenging diagnoses, provide patients with more precise prognostic information, and allow physicians to tailor therapy to a patient tumor's unique molecular signature. Additionally, for the many other aforementioned cancer types with frequent mutations in IDH1/2 and the TERT promoter at these loci, such a diagnostic tool would assist in rapid and sensitive detection of these mutations also.
Current diagnostic efforts for mutation detection are based on Sanger sequencing, which is time-consuming, costly, and most importantly is limited by poor sensitivity (limit of detection ˜20% mutant alleles)17. Samples of low tumor percentage (<40% tumor, for heterozygous mutations implies <20% mutant alleles) can be misdiagnosed as lacking mutations due to limited sensitivity (FIG. 4, 5). Such scenarios of low tumor percentage are very realistic in the context of diffuse glioma and other malignancies. In addition to the inherent heterogeneity and invasive nature of diffuse glioma, tumor biopsies can contain minimal tumor tissue, leading to poor sampling. Necrosis is a feature common to many cancers that can also limit usable tissue. These limitations have significant diagnostic and prognostic implications and can result in misguided therapy, making them inadequate for clinical use.
There is a continuing need in the field to make clinical analyses faster, more sensitive, and more specific.